The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Further, the utilization of such technology for substitution of mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.
One application of such a strategy is the “humanization” of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study the mechanisms underlying programmed expression and assembly of antibodies as well as their role in B-cell development.
Further, the strategy of humanizing a mouse humoral immune system would provide an ideal source for production of fully human antibodies, particularly monoclonal antibodies (Mabs)—an important milestone towards fulfilling the promise of antibody therapy in human disease. Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to non-human (e.g., rodent) or non-human-derivatized Mabs and thus to increase the efficacy and safety of the administered antibodies. The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as inflammation, autoimmunity, and cancer, which require repeated antibody administrations.
The strategy of humanizing a non-human transgenic animal to produce fully human monoclonal antibodies is important also because it avoids problems encountered with other methods of obtaining fully human antibodies and antibodies that have been altered to reduce adverse immunogenic effects, i.e., “humanized” antibodies. Although useful, humanizing techniques have a number of disadvantages, including labor-intensive protocols and potential alterations of specificity and/or affinity of the variable regions for the original epitope, and contamination of the variable region with residual non-human sequences that may result in host rejection. Making efficacious human monoclonal antibodies in vitro also has proven difficult. Moreover, most of the human monoclonal antibodies produced in vitro have been IgM, which is sometimes associated with immune complex formation and enhanced inflammation.
One approach towards the goal of making fully human antibodies in non-human transgenic animals is to engineer strains deficient in endogenous antibody production that produce human antibodies from large inserted fragments of the human Ig loci. Large fragments have the advantage of preserving large variable gene diversity as well as sequences necessary for the proper regulation of antibody production and expression. By exploiting the host machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the human antibody repertoire reproduced in these engineered strains includes high affinity antibodies against any antigen of interest, including human antigens. Then, antigen-specific human Mabs with the desired specificity give readily produced and selected using e.g., hybridoma technology.
The success of this general strategy was demonstrated in connection with the generation of XenoMouse® strains. See e.g., Green et al. Nature Genetics 7:13-21 (1994). The XenoMouse® strains were engineered with 245 kb and 190 kb-sized germline configuration fragments of a human heavy chain locus and a human κ light chain loci, respectively, that contained core variable and constant region sequences in yeast artificial chromosomes (YACs). Id. The human Ig-containing YACs proved to be compatible with the mouse system for both rearrangement and expression of antibodies. Moreover, the human loci substituted for the inactivated mouse Ig genes as demonstrated by their ability to support B-cell development and to generate an adult-like human repertoire of fully human antibodies.
This approach is further discussed and delineated in U.S. Pat. Nos. 5,939,598, 6,114,598, 6,075,181, 6,162,963, and 6,150,584; and in International Patent Applications WO 96/22380 and WO98/24893. See also European Patent EP 0 463 151 B1, and International Patent Applications WO 94/02602, WO 96/34096, and WO 96/33735. The disclosures of each of the above-cited patents and applications are hereby incorporated by reference in their entirety.
An alternative approach to making fully human antibodies utilizes an Ig “minilocus”. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the exogenous Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806 and 5,625,825, both to Lonberg and Kay, and GenPharm International U.S. patent application Ser. No. 07/574,748, filed Aug. 29, 1990; Ser. No. 07/575,962, filed Aug. 31, 1990; Ser. No. 07/810,279, filed Dec. 17, 1991; Ser. No. 07/853,408, filed Mar. 18, 1992; Ser. No. 07/904,068, filed Jun. 23, 1992; Ser. No. 07/990,860, filed Dec. 16, 1992; Ser. No. 08/053,131, filed Apr. 26, 1993; Ser. No. 08/096,762, filed Jul. 22, 1993; Ser. No. 08/155,301, filed Nov. 18, 1993; Ser. No. 08/161,739, filed Dec. 3, 1993; Ser. No. 08/165,699, filed Dec. 10, 1993; and Ser. No. 08/209,741, filed Mar. 9, 1994; the disclosures of which are hereby incorporated by reference. See also International Patent Applications WO 94/25585, WO 93/12227, WO 92/22645, and WO 92/03918, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), the disclosures of which are hereby incorporated by reference in their entirety.
An advantage of the minilocus approach is the rapidity with which constructs including portions of the Ig locus can be generated and introduced into animals. A significant disadvantage of the minilocus approach, however, is that, in theory, insufficient diversity is introduced through the inclusion of only small numbers of V, D, and J genes. Indeed, the published reports, including U.S. Pat. No. 6,300,129, describe B-cell development and antibody production in animals produced by the minilocus approach that appears stunted.
Accordingly, there is a need for producing non-human transgenic animals that comprise more complete Ig loci than has previously been produced to obtain transgenic animals having a substantially complete human antibody repertoire. Introduction of other Ig loci into a transgenic non-human animal may permit greater antibody diversity and would be likely to reconstitute a more complete immune repertoire of the animals. Thus, it would be desirable to provide transgenic animals stably containing more complete Ig V gene germline sequences, particularly having germline Vλ sequences. It would be additionally desirable to provide such loci against a knockout background of endogenous Ig. Animals capable of producing such a repertoire can be used to create immortalized cells, such as hybridomas, that make fully human monoclonal antibodies for both diagnostic and therapeutic purposes.